The invention relates to a method for optical detection of analyte molecules in a natural biological medium.
The optical detection of individual molecules was first described in Applied Optics, Volume 15 (1976) page 2965. In the following years this detection technology was improved up to the detection of individual fluorophores, i.e. individual fluorescent chemical groups (Chemical Physics Letters, Volume 174 (1990) page 553).
However, it has not hitherto been possible to detect individual molecules in natural biological mediums. Such mediums when excited with light show a strong background luminescence (Analytical Chemistry Volume 68 (1996) page 2270). The luminescence results from the fact that buffer substances, enzymes and other macromolecules are contained in natural biological mediums. This luminescence is particularly strong in the case of blood plasma with its approximately 100 different proteins.
The object of the invention is to provide a method with which individual or a few analyte molecules can be detected in a natural biological medium.
In order to achieve this object the generic method is characterized in that
the analyte molecules are marked with at least one fluorescent dye;
that from an observed volume single photons are absorbed in the natural biological medium in order to carry out a time-correlated single photon count and to obtain time data for the single photons;
that at least two patterns are predetermined, wherein a first pattern describes time data expected from the at least one fluorescent dye and a second pattern describes time data expected from the natural biological medium;
that a comparative model is formed by a weighted addition of the patterns;
that the comparative model is adapted to the obtained time data by variation of the weighting factors;
that the values of the weighting factors for optimum conformity of the comparative model with the obtained time data are determined; and
that the presence of at least one analyte molecule is assumed when the determined value of the weighting factor for the first pattern exceeds a predetermined threshold.
The marking of the analyte molecules with fluorescent dyes makes even non-fluorescent analyte molecules detectable.
The time data obtained by time-correlated single photon counts can be represented in the form of a decay curve for each predetermined time interval. The decay curve shows the progress of the fluorescence decay or respectively of the decay over time of the luminescence of a specimen located in the observed volume. As a rule the natural biological medium has a short luminescence decay time. For undiluted blood plasma the decay time in the case of excitation with light having a wavelength of 637 nm (1 nm=1 nanometer=10xe2x88x929 m) and detection of the photons in the wavelength range from 650 to 700 nm amounts to approximately 300 ps (1 ps=1 picosecond=10xe2x88x9212 sec). If fluorescent dyes having a substantially longer fluorescent lifetime, e.g. 4 ns (1 ns=1 nanosecond=10xe2x88x929 sec), are chosen for the marking, then it is possible to establish how greatly the fluorescent dyes have contributed to the decay curve.
This can be achieved mathematically by the addition of known decay curves, so-called patterns, for the background luminescence of the natural biological medium and the fluorescent dyes used for marking, after multiplication by weighting factors. From this a comparative model is obtained. The weighting factors are then varied, and the values of the weighting factors for optimum conformity of the comparative model with the obtained time data are determined.
The values for the weighting factors which are obtained after optimization give information as to how great the amount of photons emanating from the fluorescent dyes is relative to the detected photons. If this amount is large or if it exceeds a predetermined threshold, then there is a high probability that single or in any case a few analyte molecules are present in the observed volume.
The method according to the invention therefore facilitates a high discrimination between the fluorescence of the fluorescent dyes and the background luminescence of the natural biological medium. With the aid of the method according to the invention it is possible, against a background of approximately 20,000 photons per second, to discriminate the approximately 100 photons which emanate from a marked analyte molecule and can be detected as the analyte molecule passes through the observed volume. This discrimination is a prerequisite for the reproducible detection of individual analyte molecules in the natural biological medium.
A further development of the invention utilizes the fact that the background luminescence of natural biological mediums, and particularly that of blood plasma, noticeably decreases when excitation light with a wavelength greater than 600 nm is used for the time-correlated single photon count. The wavelengths between 630 and 670 nm are particularly suitable for this. Since the fluorescence of the dyes is always red-shifted, a wavelength range used for the detection will always be of longer wavelength than the wavelength of the excitation. If photons are preferably detected which have a wavelength between 10 and 60 um longer than the respective excitation wavelength, then a preferred detection of the photons of the fluorescent dye and an improved discrimination between background and desired signal for the detection of individual analyte molecules is achieved.
A pulsed light source, an optical measuring arrangement and a detector, connected to an electronic detection arrangement, are used in the usual way for the time-correlated single photon counts in order to be able to detect the time gap between the time of detection of a photon and the time of the excitation pulse. In a preferred further development of the invention a diode laser is used as source for the excitation light. Diode lasers are very economical, very small and generate light at the desired wavelengths in the range from 630 to 670 nm.
In an advantageous further development of the invention a natural biological medium is examined with a number of analyte molecules. The different analyte molecules are specifically marked with different fluorescent dues each having different fluorescence decay behavior, e.g. difference fluorescent lifetimes.
In the case of mono-exponential fluorescent decay curves the decay curves for predetermined time intervals are modeled in such a way that the fluorescent lifetime is treated as an additional variable to be adapted. If the detected time data are described by such a model and if the values for the weighting factors and the fluorescent lifetimes are determined by adaptation, then on the one hand the weighting factors make it possible to determine whether a single or a few analyte molecules is/are present in the observed volume. However, the determination of the optimally appropriate fluorescent lifetime also makes it possible to make a statement as to the type of fluorescent dye used. In particular in this case it is possible to establish which of a plurality of different fluorescent dyes used for marking has been detected. This permits an identification of the analyte molecules specifically coupled to the dyes.